1. Field of the Invention
The invention generally relates to the improvement of clavulanic acid production in Streptomyces clavuligerus by genetic engineering. In particular, the invention provides genetically engineered S. clavuligerus in which a newly identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is disrupted, resulting in increased clavulanic acid production.
2. Background of the Invention
The β-lactams penicillin and cephalosporin were among the first useful antibiotics discovered and remain at the forefront of clinical use to combat bacterial infections. The wide-spread use of β-lactam antibiotics for more than 50 years, however, has reduced their effectiveness owing to the emergence of resistance among invading pathogens. As a consequence, strategies aimed at overcoming acquired resistance have become of increasing interest. One of the best examples in broad clinical application is the development of β-lactamase inhibitors. The discovery of clavulanic acid was reported in 1976, (Reading and Cole, 1977) and it has been shown to be a potent inhibitor of β-lactamases produced by staphylococci and plasmid-mediated β-lactamases of E. coli, as well as species from Klebsiella, Proteus, and Hemophilus (Brown et al., 1976). The molecule is produced by the filamentous bacterium Streptomyces clavuligerus, and consists of a β-lactam ring fused to an oxazolidine ring (Howarth et al., 1976; Reading and Cole, 1977). Commercial products such as Augmentin® and Timentin®, which are combinations of clavulanic acid and other established β-lactam antibiotics, are prescribed in more than 150 countries and have attained sales in excess of 2 billion dollars yearly (Elander, 2003).
To date strain improvement of microorganisms to obtain high-titers of secondary metabolites that are more suitable for industrial fermentations has depended largely on random mutagenesis and selection techniques. However, development of a new generation of high production strains with this approach often takes 5 years or more (Nielsen, 1997). A significant drawback is the introduction of a limited spectrum of base-pair substitutions that do not readily solve the specific rate limitations of biosynthetic pathways (Baltz, 1998). In the past few years, as techniques for molecular genetics have become increasingly sophisticated, the ability to modify existing pathways or create non-native pathways has advanced rapidly. Progress in genetics, transcriptional analysis, proteomics, metabolic reconstructions and metabolic flux analysis offer genetic engineering as an alternative approach for strain improvement in a targeted manner (Baltz, 2001). Duplication of specific genes thought to be involved in rate limiting steps can be achieved by inserting the desired gene(s) into a chromosome by homologous recombination or by site-specific integration. In S. clavuligerus, gene dosage constructs of the biosynthetic genes ceas and cs2 resulted in recombinant strains with 60% and 100% higher clavulanic acid production, respectively, compared to the wild-type strain (Perez-Redondo et al., 1999). Disruption of negative regulatory gene(s), or increased expression of positive regulatory gene(s) also can result in the elevated production of secondary metabolites. Paradkar, et al. observed a 2 to 3-fold increase in clavulanic acid production by introducing additional copies of positive regulatory genes in the wild-type (Paradkar et al., 1998; Perez-Llarena et al., 1997; Perez-Redondo et al., 1998). A third approach is to inactivate pathways that compete for key precursors, intermediates, cofactors and energy supply. The inactivation of the clavam pathway, which shares the common intermediate clavaminic acid with the clavulanic acid pathway, has been shown to give an elevated yield of clavulanic acid in S. clavuligerus (Paradkar et al., 2001).
The primary metabolic precursors of clavulanic acid are D-glyceraldehyde-3-phosphate (G3P) (Khaleeli et al., 1999) and L-arginine (Valentine et al., 1993). The observation of arginase and ornithine carbamoyltransferase activities are strongly suggestive of a functional urea cycle in S. clavuligerus (Bascaran et al., 1989; Ives and Bushell, 1997; Romero et al., 1986). The prokaryotic urea cycle is unusual and provides a very effective pathway for arginine biosynthesis such that the pool size of this amino acid could remain sufficient to support an increased rate of clavulanic acid production. Supplemented fermentations of S. clavuligerus with arginine increases only the intracellular pool size of this precursor, but not the production of clavulanic acid (Chen et al., 2002; Chen et al., 2003). Metabolic flux analysis has further suggested that a limiting factor for clavulanic acid biosynthesis is the C3 precursor, G3P (Ives and Bushell, 1997). This deduction was supported by the observation of a stimulatory effect on clavulanic acid production by supplementing cultures of S. clavuligerus with glycerol (Chen et al., 2003). G3P is an intermediate of the glycolytic pathway and also the entry point in the gluconeogenesis pathway for the synthesis of glucose. Metabolic analysis has further shown that in wild-type S. clavuligerus the favored direction of G3P flux (˜80%) is consistently towards the glycolytic pathway, and the rest (˜20%) enters the gluconeogenesis and clavulanic acid pathways (Kirk et al., 2000) (see FIG. 1). G3P is converted into 1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the glycolytic pathway and finally enters the Krebs cycle through pyruvate.
While these observations suggest that increasing the intracellular pool of G3P could result in enhanced clavulanic acid production in S. clavuligerus, the prior art has thus far failed to exploit this potential, even though there is an ongoing need to develop additional strains with improved capacity to produce high yields of clavulanic acid.